Fluorescence Spectroscopy of BiologicalTissues—A Review

Luciano Bachmann

Departamento de Fısica e Matematica, Faculdade de Filosofia, Ciencias e

Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto,

Sao Paulo, Brazil

Denise Maria Zezell, Adriana da Costa Ribeiro, and Laercio Gomes

Centro de Lasers e Aplicacoes, Instituto de Pesquisas Energeticas e

Nucleares, Sao Paulo, Brazil

Amando Siuiti Ito

Departamento de Fısica e Matematica, Faculdade de Filosofia, Ciencias e

Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto,

Sao Paulo, Brazil

Abstract: Fluorescence of the skin, enamel, dentin, and bone are reviewed.

Fluorescence spectroscopy is one of the noninvasive methods that can identify

diseases and promote increasing the knowledge in medical diagnosis. The microstruc-

ture and composition of biological tissues are presented, followed by a description of

chromophores, fluorophores as identified by use of applied fluorescence techniques.

Keywords: Fluorescence, skin, enamel, dentin, bone

INTRODUCTION

Biological tissues consist of heterogeneous structures that promote light scat-

tering. They contain chromophores that absorb light, as well as fluorophores

Received 10 July 2006, Accepted 21 July 2006

Address correspondence to Luciano Bachmann, Departamento de Fısica e Matema-

tica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao

Paulo, Av. dos Bandeirantes 3900, Ribeirao Preto, Sao Paulo 14040-901, Brazil.

E-mail: bachmann@ffclrp.usp.br

Applied Spectroscopy Reviews, 41: 575–590, 2006

Copyright # Taylor & Francis Group, LLC

ISSN 0570-4928 print/1520-569X online

DOI: 10.1080/05704920600929498

575

that absorb and reemit light. The consequent tissue optical properties such as

scattering, absorption, reemission, and reflection can help us characterize the

tissue and identify diseases by noninvasive methods. In this article, several

tissues are studied (skin, enamel, dentin, and bone) and their excitation and

fluorescence peaks will be compared with the peaks observed for biological

molecules (fluorophores).

BIOLOGICAL TISSUE ARCHITECTURE AND COMPOSITION

Skin

The human skin is divided into layers. Each has a specific composition that

provides it with a characteristic optical profile (1). The outermost layer of

the skin is the stratum corneum. This layer is composed mainly of dead

cells embedded in a lipid matrix (2). The second layer, beneath the stratum

corneum, is the epidermis. In this layer, melanin absorbs a great part of the

light. Following the epidermis comes the dermis, which is composed of con-

nective tissue, nerves, and blood vessels. Under the epidermis, the hypodermis

is composed of adipose tissue.

Hard Dental Tissues

The tooth consists of four tissues: enamel, dentin, cement, and pulp tissue. The

pulp irrigates the tooth interior with nutrients originating from the blood. This

tissue is composed of blood vessels, nerves, odontoblasts, and fibroblasts. The

dentin surrounds this tissue and is recovered by the cement at the subgengival

region of the tooth and by the enamel at the supragengival region. The dentin

is structured with tubules of diameters between 1 and 5 mm; the tubules arise

at the pulp-dentine interface and belong up to the dentin-enamel interface.

They are filled with water and odontoblastic processes (cells responsible for

dentin production) occur there. The collagen molecules are oriented orthogonal

to the tubules, and hydroxyapatite crystals are inserted within the collagen

matrix. In the enamel, the crystals are bound together, forming bundles called

prisms (diameter of about 5 mm). These prisms start at the dentin-enamel

interface and extend up to the tooth external surface. The enamel organic

material and water are concentrated in the interprismatic regions, while the

prisms bulk are composed mainly of hydroxyapatite crystals.

The biological hard tissues, enamel, dentin, and bone consist of a mineral

matrix (hydroxyapatite), water, and an organic matrix (collagen and a small

fraction of non-collagen proteins, lipids, citrates, and sugars) (3) (4). The

chemical composition of the three tissues are compared in Table 1. The

enamel has a small organic matrix (1 wt%) and a major inorganic matrix

(97 wt%), while the dentin and the bone have a larger organic matrix

L. Bachmann et al.576

(respectively, 20 wt% and 25 wt%) and an inorganic matrix of about 69 wt%

for the dentin and 65 wt% for the bone.

Bone

The bone structure and composition vary between different parts of the

skeleton and with age, but some general bone features can be described (5).

Contrary to the enamel and the dentin, the cells in the bone are continually dis-

solving and forming the hydroxyapatite crystals, so that this tissue is

remodeled during life. The microscopic base structures of the bone are the

tubular elements, also called osteons, with a combination of collagen and

hydroxyapatite crystals (6).

BIOLOGICAL CHROMOPHORES

The main biological molecules that absorb the light in the ultraviolet, visible,

and near infrared spectral regions are listed in Table 2. Proteins, collagen,

Table 1. Percentage values of the organic matrix, mineral matrix, and water present

in the human enamel and the dentin tissue (4)

Dentin Enamel Bone

vol% wt% vol% wt% vol% wt%

Inorganic

matrix

47 70 87 97 36 65

Organic

matrix

30 20 2 1.5 35 25

Water 21 10 11 1.5 28 10

Table 2. Main chromophores present in biological tissues and their absorption peaks

in the ultraviolet, visible, and near infrared spectral region

Absorption peaks (nm) Chromophore Reference

412, 542, 577 Oxyhemoglobin (13) (25) (26)

430, 555, 760 Deoxyhemoglobin

Increase to short wavelengths Melanin

760, 900, 1250, 1400, etc. Water

460 Bilirubin

260 DNA/RNA

280 Urocanic acid

�290, �320 Collagen (27)

�325 Elastin

Fluorescence Spectroscopy of Biological Tissues 577

elastin, DNA/RNA and urocanic acid absorb in the ultraviolet region

(wavelength shorter than 400 nm), while oxyhemoglobin, deoxyhemoglobin,

melanin, and bilirubin absorb light in the visible region (400–700 nm). In the

infrared region (wavelength longer than 700 nm), we can observe the deoxyhe-

moglobin band (760 nm), water bands (760 nm, 900 nm, 1250 nm, 1400 nm,

etc.), and other vibrational absorption bands at higher wavelengths (not listed).

The two types of hemoglobin are found in blood cells and the high absorp-

tion band observed near 400 nm gives blood its reddish color. Melanin is

observed in the epidermis and is responsible for the skin color (7). Bilirubin

and b-carotene can be found in all skin layers: stratum corneum, epidermis,

and dermis; and the structural proteins, collagen and elastin, are found in

soft tissues as well as in hard tissues such as dentin and bone.

OPTICAL SPECTROSCOPY TECHNIQUES

Changes with age, diseases, or other cellular processes will also change tissue

properties, so these changes can be used to distinguish between these tissues

and the healthy ones. Different optical techniques have the potential to access

the tissue optical characteristics by noninvasive procedures.

When light interacts with the tissue it can be absorbed, reflected,

reemitted, or scattered. Absorbed light can be measured by ATR (attenuated

total reflection) or photoacoustic techniques; reflected light can be measured

by diffuse reflectance spectroscopy, visible-infrared images, OCT (optical

coherence tomography), or confocal microscopy; re-emitted light can be

measured by fluorescence-excitation spectroscopy, two-photon microscopy,

or confocal microscopy; and scattered light can be measured by scattering

spectroscopy or Raman spectroscopy.

When molecules are stimulated by light in the cells, they respond by

becoming excited and can thus re-emit light of varying wavelengths, which

can be measured. Just as a prism splits white light into a full color

spectrum, laser light focused on the tissue can be reemit in colors determined

by the properties of the molecules and its environment.

FLUORESCENCE SPECTROSCOPY TECHNIQUES

While it is beyond the scope of this review to fully describe fluorescence spec-

troscopy, the following will provide an overview for the readers. Detailed

description is to be found elsewhere (8).

Usually simplified diagrams such as that presented in Figure 1 are used to

represent the energy levels of a molecule. These energy levels include the

ground electronic state (S0) and higher energy electronic states (e.g., S1, S2)

reached upon the absorption of light and are represented by thick lines.

Each electronic state of a molecule also contains numerous vibrational and

L. Bachmann et al.578

rotation energy levels that fully describe the energetic of the system and is

represented by thin lines in the figure.

The absorption of a photon with energy hvA excites the fluorophore from

its electronic ground state (S0) to upper electronic states (S1, S2, . . .). The exact

vibrational and electronic level reached will depend upon the energy content

of the light absorbed. Regardless of the excited level reached, the molecule

will rapidly lose energy to its environment through non-radiative modes

(internal conversion) and will revert to the lowest vibrational level of the

lowest electronic excited state. The transition from this state to the ground

state may be accompanied by the emission of a photon with energy hnF in

the process called fluorescence emission. The molecule may persist in this

lowest level of the S1 state for a period of time known as the fluorescence

lifetime, which, for most fluorophores of interest in tissues, are in the range

of several nanoseconds to a few tens of nanoseconds.

The main components for a fluorescence spectroscopy instrument are

represented in Figure 2. The excitation source can be lamps (deuterium,

Figure 1. Schematic representation of a fluorophore energy levels.

Figure 2. Schematic illustration of the instrumentation for fluorescence

spectroscopy.

Fluorescence Spectroscopy of Biological Tissues 579

Table 3. Fluorescence peaks of soft tissue samples

Excitation

(nm) Florescence peaks (nm) Sample description Reference

270 300 315 325 350 Type I acid-soluble collagen (0.05% in 0.5 M acetic acid) (28)

350 430

337 390 460 Normal colonic tissue (29)

390 460 630 680 Hyperplastic polyps; adenomatous polyps

394 Collagen type I

— 385 460 Aging human insoluble collagen-rich tissue (9)

370 440 Collagen from rat and human tissue (10)

337 380 Type I collagen (achilles tendon) (19)

420 Type I collagen (calf skin)

400 Type I collagen (rat tail)

380 Type II collagen (bovine tracheal cartilage)

380 Type II collagen (bovine nasal septum)

385 Type III collagen (human placenta)

410 Type IV collagen (human placenta)

405 Type V collagen (human placenta)

280 410 Skin (30)

325 405

365 440 Rat tail tendon (31)

330 �427

480 �540

510 �560

570 �620 Native collagen (32)

630 �670

680 �690

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xenon, tungsten), light-emitting diodes (LEDs) or lasers systems. If the source

is not monochromatic, like the lamps, additional components such as mono-

chromators or filters must be used to select the desired excitation wavelength

or range of wavelengths.

After selecting the excitation wavelength, the beam can be handled using

lens, mirrors, or fibers to irradiate the sample. The emitted light from the

sample will be selected by a monochromator and detected through photo-

diodes, charge-coupled devices (CCDs), InGaAs detectors, or photomulti-

pliers. Usually the emission beam is examined in a direction that makes an

angle of 908 from the excitation beam direction, in the so-called L

geometry, to avoid detection of transmitted light. Emission (or excitation)

spectra are obtained fixing the excitation (or emission) wavelength and

measuring the intensity of the light coming from the sample as a function

of the emission (or excitation) wavelength.

FLUORESCENCE OF SOFT TISSUES

Fluorescence peaks of soft tissues are summarized in Table 3 and the exci-

tation peaks in Table 4. Fluorescence peaks of normal soft tissues occur in

Table 4. Excitation peaks of soft tissue samples

Fluorescence

(nm) Excitation peaks (nm)

Sample

description Reference

360 265 280 Type I acid-sol-

uble collagen

(0.05% in

0.5 M acetic

acid)

(28)

435 350–360

— 335 360 Aging human

insoluble col-

lagen-rich

tissue

(8)

440 370 Collagen from

rat and human

tissue

(9)

— 295 335 350 370 Human skin

(27-year-old

volunteer)

(13)

295 335 350 370 380 420 Human skin

(70-year-old

volunteer)

350 264 Skin (30)

410 322

Fluorescence Spectroscopy of Biological Tissues 581

Table 5. Fluorescence peaks of natural and carious hard tissues

Excitation

(nm) Fluorescence (nm) Sample description Reference

280 407 Natural dentine (30)

325 413

365 440 590 640 Whole bone;

collagen and

apatite extracted

from bone

samples

(31)

375 460 560 EDTA dissolved

human dental

enamel

(33)

460 560 EDTA dissolved

synthetic

hydroxyapatite

(33)

250–320 397–402 Sound human

dentin

(34)

285 355 Fluorophores

extracted from

normal dentin

with HCl

(35)

350 410–440

280–370 350 405 450 520 Solid human/bovine enamel

(36)

360 410 455 Organic com-

ponent extracted

from enamel

295 360 400 Hydrolyzed

enamel and

dityrosine

(37)

365 430–450 Natural dentin (11)

480 550 Carious and non-

carious enamel

(38)

337 400 Carious and non-

carious enamel

(33)

488 540

407 590 625 635 700 Carious enamel

and dentin

(16)

250–320 425 Carious human

dentin

(34)

400 480 624 635 690 Carious enamel (39)

400 480 624 650 687 Root carious (40)

405 455 500 582 622 Sound and carious

enamel

(41)

337 440 490 590 630 Carious enamel (42)

(continued )

L. Bachmann et al.582

the spectral region between 300 nm and 460 nm, and the excitation peaks

occur in the region between 265 nm and 370 nm. During aging of human

collagen in the skin tissue, an increase in the fluorescence and excitation

peaks occurs (9, 10). The collagen excitation (350 nm) and fluorescence

(430 nm) maxima increase significantly between young (age 19) and old

(age 81) humans. An absorption band is also observed in the ultraviolet

region (250–400 nm) (11, 12) and additional excitation peaks are observed

at 380 nm and 420 nm in old human skin (13).

FLUORESCENCE OF HARD TISSUES

Wavelength of fluorescence peaks of natural hard tissues are summarized in

Table 5 and of the excitation peaks in Table 6. The major fluorescence

peaks of natural tissues occur at the spectral region between 350 nm and

560 nm, and the excitation peaks between 268 nm and 375 nm. The fluor-

escence and excitation peaks of carious tissues occur at longer wavelengths:

fluorescence in the region between 540 nm and 700 nm, and excitation in

the region between 398 nm and 632 nm. In general, it is possible to observe

that after caries attack, the fluorescence and excitation peaks change to wave-

lengths of lower energy.

AMINO ACIDS

As the fluorescence persists after collagen degradation (14), the natural

collagen fluorescence must arise from fundamental units of this molecule

Table 5. Continued

Excitation

(nm) Fluorescence (nm) Sample description Reference

337 405 435 490 525 Sound and tooth (43)

405 435 490 555 Dentin level caries

405 435 490 530 635 Pulp level caries

420 495 595 635 695 Dental calculus

(supragingival)

(44)

420 495 595 650 695 Dental calculus

(subgingival)

635 700 783 Dental calculus

(subgingival)

(45)

655 720 810 Carious dentine (45)

405 500 Sound and carious

enamel

(46)

Fluorescence Spectroscopy of Biological Tissues 583

Table 6. Excitation peaks of natural and carious hard tissues

Fluorescence

(nm) Excitation (nm) Sample description Reference

350 268 Natural dentine (30)

410 324

320 285 HCl dissolved normal

dentin

(35)

350–460 285 335 375 Solid human/bovine

enamel

(36)

285 330 Organic component of

enamel

360 285 315 Hydrolysed enamel (37)

700 400 466 505 540 582 632 Carious lesion (enamel/dentin)

(16)

635 400 465 White spot lesions (39)

624 405 465 Light brown; dark brown

lesions

633 398 405 507 538 Dental calculus

(supragingival)

(44)

700 402 412 506 577 628 Dental calculus

(subgingival)

(45)

700 394 527 629 Carious dentine (45)

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such as amino acids or peptides. Dentin and skin fluorescence peaks are

compared with different amino acids and peptides fluorescence peaks in

Table 7 (15). For the amino acids, only hydroxylysine and tyrosine display

emission near the collagen fluorescence peak (410 nm). Certain peptides

like glycyl-aspartic acid and glycyl-serine also display a peak near 410 nm.

Other amino acids exhibit fluorescence not exactly at 410 nm, but near this

wavelength, so they can also contribute to the final collagen fluorescence

observed in the case of the dentin and the skin. To summarize, the main

fluorophores present in soft and hard tissues are listed in Table 8.

PORPHYRINS

The fluorescence peaks of various porphyrins are summarized in Table 9 (16)

and also compared with microorganisms fluorescence peaks (17). Porphyrins

are products of microorganisms and can be one of the endogenous fluor-

escence observed in dental caries. In the literature, the caries fluorescence

peak at 635 nm is attributed to protoporphyrin IX, the 625 nm peak to

coproporphyrin, and the 590 nm peak to Znproto- porphyrin (16). The main

microorganisms responsible for the plaque flora are Streptococci,

Table 7. Fluorescence peaks at two fixed excitation wave-

lengths of various amino acids and peptides (15); the peaks

are compared with dentin and skin samples

Excitation (nm)

280 325 Amino acids/peptides

Fluorescence (nm)

360 367 Tryptophan

— 410 Hydroxylysine

321 — Phenylalanine

325 — Histidine

312 414 Tyrosine

408 410 Tetraglycine

414 413 Triglycine

410 405 Glycyl-aspartic acid

415 410 Glycyl-serine

432 451 Histidyl-histidine

400 416 Glycyl-asparagine

— 408 Glycyl-proline

— 420 Glycyl-prolyl-glycyl-glycine

410 410 Dentin

410 410 Skin

Fluorescence Spectroscopy of Biological Tissues 585

Actinomyces, and Bacteroides. No typical fluorescence in the red spectral

region was found in the case of bacterial strains Streptococcus mutans and

Lactobacterium. Fluorescence was observed only in the case of Actinomyces

odontolyticus, Bacteroides intermedius, Pseudomonas aeruginosa, Candida

albicans, and Corynebacterium (17).

Table 8. Assignment of the main fluorophores present in soft and hard tissues

Excitation

peak

(nm)

Fluorescence

peak (nm) Fluorophores Reference

270 320 Tyrosine (47)

295 345 Tryptophan

335/370 390/460 Collagen cross-links

420/460 500/540 Elastin/collagen cross-links (13)

405 600 Porphyrins

350 460 NAD/NADH

370 460 Keratin, horn

280 350 Tryptophan (27) and

references

cited

therein

275 300 Tyrosine

260 280 Phenylalanine

325 400, 405 Collagen

290, 325 340, 400 Elastin

450 535 FAD (flavin adenine

dinucleotide), flavins

290, 351 440, 460 NADH (reduced nicotinamide

adenine dinucleotide)

336 464 NADPH (reduced nicotinamide

adenine dinucleotide

phosphate)

327 510 Vitamin A

335 480 Vitamin K

390 480 Vitamin D

332, 340 400 Pyridoxine

335 400 Pyridoxamine

330 385 Pyridoxal

315 425 Pyridoxic acid

330 400 Pyridoxal 5-phosphate

275 305 Vitamin B12

436 540, 560 Phospholipids

340–395 540, 430–460 Lipofuscin

340–395 430–460, 540 Ceroid

400–450 630, 690 Porphyrins

270–280 360 Excimer-like species (32) (48)

325 400 Dityrosine (49)

370 450 Species observed in

collagen of old humans

(48)?

L. Bachmann et al.586

FLUORESCENCE LIFETIME

Fluorescence lifetime helps distinguish between two or more compounds that

emit at similar wavelengths. The fluorescence decay time of natural hard

Table 9. Fluorescence peaks of various porphyrins in solutions

(16) and microorganisms that can be found in dental caries (17).

The spectra were recorded at a 407 nm excitation wavelength

Fluorescence peaks (nm) Sample description

Porphyrins

633 700 Protoporphyrin IX

623 690 Coproporphyrin

593 646 Zn-protoporphyrin

Microorganisms

636 708 Actinomyces odontolyticus

635 708 Bacteroides intermedius

618/635 703 Pseudomonas aeruginosa

620 �700 Candida albicans

600 �680 Corynebacterium

Table 10. Fluorescence lifetime of natural hard dental tissues, carious enamel, and

different collagen types of soft tissues

Lifetime (ns) Sample description Reference

0.1–0.2; 5.7–6.3;

17.5–19.0

Natural dentine (11)

0.5 (15%); 3.18

(46%); 9.76 (39%)

Natural enamel (18)

0.31 (7%); 2.27

(11%); 17.25

(82%)

Carious enamel

20 (100%) Coproporphyrin (18)

3 (11%); 17 (89%) Protoporphyrin

2 (92%); 13 (8%) Zn-protoporphyrin

5.2 Type I collagen (achilles tendon) (19)

1.05 Type I collagen (calf skin)

1.45 Type I collagen (rat tail)

6.1 Type II collagen (bovine tracheal

cartilage)

6.2 Type II collagen (bovine nasal septum)

2.95 Type III collagen (human placenta)

1.25 Type IV collagen (human placenta)

1.05 Type V collagen (human placenta)

Fluorescence Spectroscopy of Biological Tissues 587

dental tissues, the carious enamel, and soft tissues are summarized in Table 10.

Both the enamel and the dentin exhibit a fluorescence spectrum consisting of

three different fluorescence lifetimes (11, 18). In the carious enamel it is

possible to observe an increase in those lifetimes. As protorphyrin IX is con-

sidered one possible source of the carious fluorescence (18), in the same table

we compare the fluorescence lifetime of different porphyrins. In soft tissues,

the different collagen types (Table 10) have fluorescence with lifetimes

roughly between 1 ns and 6 ns (19), similar values to that of the three

lifetimes observed in dentin. Another group of molecules, the metal-free

porphyrin monomers, have a long fluorescence lifetime, of about 10–20 ns

(20–23). In contrast, most of the endogenous fluorophores like the fluorescent

coenzymes NADH and flavin molecules or the amino acid tryptophan have

lifetimes shorter than 6 ns (24).

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